At present, millions of coronary patients throughout the world are treated with digitalis glycosides [J. R. Ochs, G. Bodem, Med. Welt 30, 602 (1978)]. Thus, this group of preparations are among the drugs most frequently prescribed. Within the entire group, digoxin plays the most important part, having a share of over 90%.
However, the wide distribution and frequent use of the digitalis glycosides should not conceal the fact that these are potentially dangerous substances-their therapeutic range is extremely small. Therefore, in order to achieve an effective and safe therapy, it is essential to monitor the digitalis level continuously. A series of different test procedures have been developed for this purpose wherein the antibodies formed against the glycoside by the body are used as test reagents. These antibodies are obtained from the serum of host animals immunized with digitalis. In this way, antisera of a polyclonal nature are obtained, that is, these sera contain different antibodies.
A major problem, however, in a number of tests for digoxin is the strong cross-reaction with digitoxin, a substance which differs from digoxin only in the presence of an hydroxyl group in 12-position: ##STR1## Structural formulas of (1) digoxin, (2) digitoxin and (3) spironolactone.
However, even more important than a cross-reaction caused by digitoxin, is the possibility of cross-reactive interference, caused by spironolactone (for example, Aldactone.RTM.), an aldasterone antogonist which is frequently administered with digoxin, which occurs in many assays, even commercial ones. This substance is administered in a substantially higher dosage, and if the test is not sufficiently discriminating, it can simulate dangerously high levels.
Attempts have been made, by laborious procedures for purification by affinity chromatography, to improve these cross-reactivities of the antisera which are conventionally produced in rabbits or sheep. However, a disadvantage encountered here is the need for large quantities of antiserum (on account of low purification yields) and the fact that any improvement in the cross-activity is often accompanied by a significant reduction in the detection sensitivity. This is due to the fact that these particular high-affinity antibodies which are responsible for a high sensitivity are very difficult to elute, or even impossible to elute, from the affinity matrix.
As an alternative to the conventional methods of producing antibodies, since the end of the seventies the production of antibodies by hybridoma cell lines in cell culture has become increasingly important, based on the pioneering work of KOHLER and Milstein 1975/76 [Nature, 256, 495 (1975)]. These cell lines are obtained after somatic fusion of spleen cells from a previously immunized mouse with cells of a mouse tumor line and subsequent repeated cloning steps.
Since they can all be traced back to a single parent cell, they are distinguished by the fact that they each produce exclusively only a single type of antibody of uniform specificity, that is, monoclonal antibody (mAK). Moreover, their theoretically unlimited growth as a tumor cell line makes it possible to produce theoretically unlimited quantities of antibody.
For tests for continuously monitoring digoxin-treated patients, or for clearing up cases of poisoning, it is sensible and desirable to use an mAK in the test system precisely because of the typical properties mentioned above.